Significant efforts are being directed toward design of novel vaccines and antivirals to confront an influenza pandemic threat. Moreover, emergence of drug resistant mutants, including those dually resistant to existing drugs M2-blockers and neuraminidase (NA) inhibitors has been reported. High-throughput screening assays are essential tools for identification of antivirals with novel and as-yet-unidentified mechanisms of action. Here we propose to develop such an assay with the use of the genetically engineered influenza virus encoding the luciferase instead of NA (delNA/luc-mutant) and MDCK cell line, which allows the NA-independent virus growth. The antiviral effect will be evaluated based on reduction of luminescence in the delNA/luc-mutant-infected cells. Previously, we generated the influenza virus encoding eGFP and used it to monitor the virus spread in live cells. The mutant did not require NA activity for production of infectious yields in cell culture and yet was highly attenuated in vivo. With the use of the reverse genetic technique, we have recently generated viruses carrying the modified the delNA-gene that encodes either Firefly or Renilla luciferase. Both enzymes are monomeric and small (61kDa and 36kDa), and neither requires post-translational processing. To measure the enzyme activity, we took advantage of the Dual-Glo Luciferase Assay kit (Promega), which was designed to allow robotic high-throughput analysis of cells containing genes of Firefly and Renilla luciferases grown in 96- or 384-well plates. The reagent was added directly to the delNA/luc-mutant-infected cells in growth medium without washing or preconditioning. The luminescence was measured with the use of Victor3 plate reader (PerkinElmer), the instrument that could be equipped with a plate stacker, a shaker, dispenser modules, an Enhanced Security mode, and other options for easy integration into a robotic system. Having shown the proof-of-principal, we now plan 1) to identify the optimal coding strategy for luciferase; 2) to evaluate the chimeric gene stability; and 3) to assess the effects of various parameters (e.g., cell culture seeding density, M.O.I., time p.i., medium volume etc.) on the outcome of the readings. The assay is versatile, and modifications can be made to generate the luciferase-encoding mutants susceptible to the existing drugs, including NA inhibitors. Because the luciferases use different substrates, the assay also permits evaluation of antiviral activity against two viruses at once (i.e., H1 and H5). [unreadable] [unreadable] [unreadable]